Volume 107, Issue 8, Pages 1810-1820 (October 2014) Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs  Julia Peukes, Timo Betz  Biophysical Journal  Volume 107, Issue 8, Pages 1810-1820 (October 2014) DOI: 10.1016/j.bpj.2014.07.076 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 Experimental procedure: (A) sketch of the setup, where the laser position is controlled by an acousto-optical deflector (AOD) and imaged in the back focal plane of the objective 1 (Obj1) via a telescope formed by the lenses L1 (f = 15 cm) and L2 (f = 30 cm) and the dichroic mirror (DM). After interaction with the bleb (see inset) the light is collected by the objective 2 (Obj2) and the back focal plane of Obj2 is imaged to the QPD via L3 (f = 9 cm) and L4 (f = 4.5 cm). (B) Example of the experimental procedure, where a bleb is identified and the position and direction of the scan line is controlled by the experimenter via a custom-programmed user interface (LABVIEW; National Instruments, Austin, TX). Scale bar, 5 μm. (C) Example of three scan results at different time-points. The edge position is defined by the first scan and used in the following sequence to determine the edge movement. (D) Measured time evolution of the bleb membrane during bleb growth and arrest. Biophysical Journal 2014 107, 1810-1820DOI: (10.1016/j.bpj.2014.07.076) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 To calculate the effective tension from the fluctuations of the bleb edge, we explored two different methods that both rely on a separation of timescales. (A) The power spectrum of the edge movement for the data shown in panel B) is calculated, and the power law of −1 is detected in the high-frequency part. The prefactor for this region of the PSD depends on the effective tension. (B) The edge movement x(t) (blue) is averaged to determine the bleb extension xbleb (red). The membrane fluctuations are recovered as the difference between edge movement and bleb extension xfluc = x − xbleb (black). (Inset) Using the determined fluctuations, we construct a position histogram and fit a Gaussian distribution to it. The variance of the Gaussian is related to the tension of a free membrane. (C) To determine the smoothing window size, the SD of xfluct(τ) is determined as a function of τ. (Black) Directly determined SD. The long time slope is determined and subtracted from the original function to obtain the corrected SD (red). The idea of the correction is to remove the effect of the continuous growth on the SD. The maximum of the resulting function is used as an estimate for the window size. (Blue dotted line) Timescale for the smoothing in this example. Biophysical Journal 2014 107, 1810-1820DOI: (10.1016/j.bpj.2014.07.076) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 (A) Direct comparison between the time-dependent edge position (black) and velocity (green). The decay of the correlation is given in the autocorrelation plot of Fig. S3 in the Supporting Material. (B) Tension (bleb panel A) resulting from the VAR method (blue) and the PSD method (red) is presented. The tension measured by different methods is consistent and suggests a systematic increase over time. The growth phase (light-brown background) is defined by the growth arrest. (C) Scatter plot of the data presented in panel B, where the markers are color codes for the edge velocity. (D) Histogram of the velocities found in the data shown in panel A. This is used to determine whether there are different phases during bleb growth that are marked by different velocities. In this example, we can identify three different growth phases that are indicated by the three peaks at positive velocities. (E) Distribution of the measured tension for all blebs and all the time-points. The distribution shows a large variation starting at 5 pN/μm up to almost 600 pN/μm. Again, the different methods show consistent values. (F) To finally check whether the two measurements are indeed consistent, we also analyzed the data using a scatter plot, where all obtained experimental values are shown. Again, the edge velocity was used as the color coding. This plot confirms that the two methods provide consistent values, inasmuch as most points lie on the diagonal (dashed line). The highest tensions typically correspond to the low velocity. The color bar of panels C and F is equivalent. Biophysical Journal 2014 107, 1810-1820DOI: (10.1016/j.bpj.2014.07.076) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 (A) Direct comparison of the growth and the stop phase. The average tension in these phases increases, whereas the tension that is found for CD-treated cells decreases significantly. (B) Plotting the normalized velocity over the measured tension shows a clear inverse relation in the log-log plot. Here we limit the data to blebs that showed at least a twofold increase of tension during measurement. This was the case for 7 out of 17 blebs (41%). Each color corresponds to a different dataset. (Dashed diagonal) Power law of −1. The same data using the VAR method can be found in Fig. S4. Biophysical Journal 2014 107, 1810-1820DOI: (10.1016/j.bpj.2014.07.076) Copyright © 2014 Biophysical Society Terms and Conditions